sin

lp

lineradonewvers aspects such as the biotechnical pre-treatment of gold ores and concentrates,abilixi

how long-term declining trendsuality of gold deposits continuesses whw in imal to traan be aess usinat foul. Microlphide

oresmaking the goldmore accessible to leachi

gold by forming gold-rich complexes and/or colloids (Reith et al.,

ore bodies (Brehm et al., 2005; Burford et al., 2003; Ehrlich, 1998;

possible activities of microorganisms is important, especially whenconsidering leaching applications, where the control of operational

Many gold deposits are sulphidic in nature and contain gold in a

ering the gold amena-yanidation) (Bosecker,

Hydrometallurgy 142 (2014) 7083

Contents lists availab

Hydrome

j ourna l homepage: www.e lse(Fig. 2) (Reith et al., 2007a). Additionally, microorganisms can inuencegold solubilisation indirectly by enhancing the permeability of

1997).The oxidation of the sulphidematrix is based on the activity of acido-

philic chemolithotrophic iron and sulphur-oxidising microorganismsconsuming the ligands that have bound gold, or by biosorption, enzy-matic reduction and precipitation, and by using gold as a micronutrient

gold particles from the sulphide matrix and rendble to dissolution using lixiviants (for example c2007a). The solubilisation of gold can be facilitated by biologicallyproduced amino acids, cyanide and thiosulphate (Reith et al., 2007a).Moreover, microorganisms can participate in the redox cycling ofiodine (Amachi, 2008), which is a potential alternative lixiviantfor gold leaching. Microorganisms can also decrease gold solubility by

form that is inaccessible to lixiviants. Refractory gold ores often containnely disseminated gold particles encapsulated by a sulphide mineralmatrix containing arsenopyrite, pyrite and pyrrhotite (Bosecker,1997). The inaccessibility of gold to lixiviant has been overcome bybiooxidising the sulphides contained in the ore, thereby liberating Corresponding author: Tel.: +61 8 9333 6253.E-mail address: anna.kaksonen@csiro.au (A.H. Kakson

1 Present address: Rio Tinto Technology and InnovationVIC 3083, Australia

0304-386X/$ see front matter 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.hydromet.2013.11.008ng by chemical lixiviants.are capable of stabilising 2.1.1. Principles of biooxidationMicroorganisms can also excrete ligandswhichGold (Au) ore grades in Australia sover time (Mudd, 2009; Fig. 1). As the qto decrease, it is expected that procestract gold from low grade ores will groindustry. Biotechnology has the potentireserves into resources. Bioprocessing cgold ores that are too expensive to procand 2) ores that contain impurities thequipment (e.g. arsenic in gold ore)gold solubilisation by oxidising the suich can economically ex-portance to the mineralsnsformuneconomic goldttractive for: 1) lowgradeg conventional processesconventional processingorganisms can mediatematrix of refractory gold

conditions may be challenging. This literature review aims to identifymicrobial processes which may be relevant or hold potential for theprocessing and recovery of gold.

2. Biotechnical pre-treatment of refractory gold ores

2.1. Biooxidation of refractory sulphide oresJongmans et al., 1997; Kumar and Kumar, 1999). Understanding the

1. IntroductionThe role of microorganisms in gold proces

Anna H. Kaksonen a,, Bhavani Madhu Mudunuru a, Raa CSIRO Minerals Down Under Flagship, Underwood Avenue, Floreat, WA 6014, Australiab CSIRO Minerals Down Under Flagship, Waterford, WA 6152, Australia

a b s t r a c ta r t i c l e i n f o

Article history:Received 9 June 2013Received in revised form 19 October 2013Accepted 13 November 2013Available online 4 December 2013

Keywords:GoldBiooxidationBioprocessLeachingPermeability

With a projected steady decmetal extraction from low-gmay provide one such solutiand indirect ways. This reviand recovery. The review comicrobially catalysed permecomplexation with biogenicen)., 1 Research Avenue, Bundoora

ights reserved.lity enhancement of ore bodies, gold solubilisation through biooxidation andviants, and microbially mediated gold recovery and loss from leach liquors.

2013 Elsevier B.V. All rights reserved.g and recoveryA review

h Hackl b,1

of gold ore grade in mineral resources, mining applications enabling efciente ores are of increasing interest to the minerals industry. Microbial processessince they can participate in the biogeochemical cycling of gold in many directexamines current literature on the role of microorganisms in gold processing

le at ScienceDirect

tallurgy

v ie r .com/ locate /hydrometwhich obtain energy by oxidising ferrous iron (Fe2+) to ferric iron(Fe3+) or elemental sulphur (S0) or other reduced sulphur compoundsto sulphuric acid (H2SO4) (Sand et al., 1995):

4Fe2 O2 4H4Fe3 2H2O 1

2S0 3O2 2H2O 4H 2SO42 2

Fe3+ and H+ ions attack the valence bonds of sulphide mineralsleading to the breakdown of sulphide matrix as shown below for pyrite(FeS2) and pyrrhotite (Fe1 xS) as examples (Belzile et al., 2004;Morin,1995; Nagpal et al., 1994).

FeS2Au 2Fe33Fe2 2S0 Au 3

FeS2 Au 14Fe3 8H2O 15Fe22SO42 16H Au 4

2Fe1xSAu 41xH 1xO221xFe2 2S0 21xH2O 2Au 7

Microorganisms used in biooxidation processes include mesophilicbacteria, such as iron- and sulphur-oxidising Acidithiobacillus(At.) ferrooxidans, sulphur-oxidising At. thiooxidans, iron-oxidisingLeptospirillum (L.) ferrooxidans and L. ferriphilum, moderately thermo-philic bacteria, such as iron- and sulphur-oxidising Sulfobacillus spp.and sulphur-oxidising At. caldus and a variety of archaea includingmesophilic iron-oxidising Ferroplasma acidiphilum, moderately thermo-philic iron-oxidising Acidiplasma cupricumulans, and thermophilicAcidianus spp., Metallosphaera spp. and Thermoplasma-like species(Bosecker, 1997; Brierley and Brierley, 2001; Golyshina et al., 2009;Hawkes et al., 2006; Olson et al., 2003; Reith et al., 2007b; Schippers,2007; van Hille et al., 2013).

In general biooxidation of the gold-containing sulphide ores is a pre-treatment which can decrease the consumption of lixiviant for goldsolubilisation in subsequent parts of the operation and ultimatelyincrease gold yields. However, since it does not actually solubilise goldbiooxidation needs to be used in conjunction with other methods.

2.1.2. Engineering applicationsDuring the past 20 years bio-treatment of refractory gold ores has

been developed as an industrial application and applied commerciallyin bioreactors and heaps. The development of the biooxidation tech-nology has been well reviewed elsewhere (see e.g. Brierley, 2008;Harvey and Bath, 2007; Ndlovu, 2008; Rawlings et al., 2003; vanAswegen and Marais, 2001van Aswegen and van Niekerk, 2004; vanAswegen et al., 2007) and hence will be only briey mentioned here.

0

10

20

30

40

50

60

1850 1875 1900 1925 1950 1975 2000 2025

Gol

d gr

ade

(g A

u t-1 )

Year

Fig. 1. Gold ore grades over time in Australia (data from Mudd, 2009). 2014 CSIRO. AllRights Reserved.

71A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083Fe1xSAu 22xFe333xFe2 S0 Au 5

Fe1xS Au 82x Fe3 4H2O 93x Fe2 SO42 8H Au 6

Primary gold ore or concentrate

Oxidised ore or concentrate

Biooxidationof sulphide minerals,

deactivation of carboneous material

and permeability enhancement

Pre-treatment of ores and

concentratesGold complexes in solution

Concentrated gold

Biooxidationand complexation

of gold

Reduction and precipitation of gold

Gold solubilisation

Recovery / loss of gold

Fig. 2. Potential roles of microorganisms in gold processing and recovery (Thiosulphate production

Organic acid production

Micronutrition

Cyanide production

Iodide production

Biosorption

Ligand utilisation and loss

Enzymatic reduction

Physical forcesThe rst industrial scale plant was started at the Fairview Mine, SouthAfrica, in 1986 (Bosecker, 1997; Morin, 1995) (Figs. 34). Since then,biooxidation operations have been commissioned in a number ofcountries, such as Australia, Brazil, Ghana, Peru, China, Uganda, USA,Kazakhstan, Uzbekistan and Russia (Table 1).

Oxidation of Fe2+ and reduced sulphur compounds

Carbon-adsorbable blanking agent

Production of acids, bases or ligands

Bioreduction of Fe3+adapted from Reith et al., 2007a). 2014 CSIRO. All Rights Reserved.

Fig. 3. BIOX process for biooxidation of refractory gold ore concentrate at the Fairview Gold mine, South Africa. 2014 CSIRO. All Rights Reserved.

72 A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083Biooxidation treatment in tank reactors is typically practised for highvalue otation concentrates (Brierley and Brierley, 2001). A number ofcommercial bioreactor processes have been developed, such as BIOX,BacTech, BACOX and BIONORD (Table 1) (Adamov et al., 2011;Brierley, 2008; Brierley and Brierley, 2001). A typical BIOX tankbiooxidation plant consists of six bioreactors congured as three prima-ry reactors operating in parallel followed by three secondary reactorsoperating in series (van Aswegen et al., 2007). This design increasesthe efciency of sulphide oxidation by reducing the short-circuitingof sulphide particles. Typically the bioreactors operate with 1520%slurry density (Olson et al., 2003). Pulp residence time in the bioreactorsis typically 46 days depending on the oxidation rate achieved,sulphide-S content and mineralogical composition of the concentrate(van Aswegen et al., 2007). Nutrients in the form of nitrogen, phospho-rus and potassium salts are added to the primary reactors to promotemicrobial growth (van Aswegen et al., 2007). The bioreactors are aeratedto maintain a dissolved oxygen concentration of N2 mg L1. As the oxi-dation of sulphide minerals is an exothermic process, the reactors arecooled continuously. Aminimumcarbonate content of 2% in theotation

concentrate is usually required to ensure that sufcient carbon dioxide

Thickened gold concentrate

Primary bioreactors

Nutrients

Mixer

Secre

Air

Lime neutralisation

To tailings dam

Process water

Fig. 4. A simplied ow sheet of the BIOX process for biooxidation of refractory gold ore cis available to promote microbial growth. If no carbonate is present,limestone or carbon dioxide enriched air can be added to the primaryreactors as a carbon source for microorganisms (Astudillo and Acevedo,2009; van Aswegen et al., 2007). Moreover, the addition of organiccarbon sources has beenproposed to promote the growth ofmixotrophicmicroorganisms (Muravyov and Bulaev, 2013). The oxidation of pyriteproduces acid, while the dissolution of carbonate minerals consumesacid. ThepHof thebioreactors is controlledwith limestone and sulphuricacid within the optimum range of 1.21.8 (van Aswegen et al., 2007).Before cyanide leaching the oxidised concentrate is typically washedin three-stage counter current decantation (CCD) circuit to removedissolved iron in order to promote gold recovery and reduce cyanideconsumption (van Aswegen et al., 2007). In some bioreactor operationsbiooxidised concentrate is directly aerated and neutralised to precipitateiron prior to cyanidation instead of solidliquid separation and washingin counter-ow decantation thickeners (Adamov et al., 2011). Othervariations include a cyanide process before biooxidation, the use ofvarious temperatures and microbial communities in consecutive stages,the use of high temperature (80 C) ferric leaching before biooxidation,

and the concentration of biomass from solutions and recycling it back

ondary actors

Tertiary reactors

Biooxidisedconcentrate to

cyanide leaching

Air Air

Counter-flow decantation thickeners Wash

water

oncentrate at the Fairview gold mine, South Africa. 2014 CSIRO. All Rights Reserved.

Table 1Examples of commercial biooxidation plants for gold recovery.

Mine Country Process Design capacity (ore t d1) Years in operation References

Fairview South Africa Reactor (BIOX) 55 1986present 1, 2, 4, 11So Bento Brazil Reactor (BIOX) 380 1991present 1, 2, 4, 5Harbour Lights Australia Reactor (BIOX) 40 19921994 1, 2, 4Wiluna Australia Reactor (BIOX) 158 1993present 1, 2, 4Ashanti-Shansu Ghana Reactor (BIOX) 960 1994present 1, 2, 3, 4Youanmi Australia Reactor (BacTech) 120 19941998 1, 2, 4Tamboraque Peru Reactor (BIOX) 60 19982003 (restarted in 2006) 1, 4, 11Beaconseld Australia Reactor (BACOX) 68 2000present 2, 4, 12Laizhou China Reactor (BACOX) 100 2001present 2, 4, 12Olympiada Russia Reactor (BIONORD) 1000 2001present 13Suzdal Kazakhsan Reactor (BIOX) 196 2005present 11Fosterville Australia Reactor (BIOX) 211 2005present 11Bogoso Ghana Reactor (BIOX) 750 2006present 11Jinfeng China Reactor (BIOX) 790 2006present 11Kokpatas Uzbekistan Reactor (BIOX) 1069 2008present 11

,40050

gs et., 20

73A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083to the bioreactors (Adamov et al., 2011; Fomchenko et al., 2010;Muravyov and Bulaev, 2013; Zaulochnyi et al., 2011). The use of variousby-products and/or waste materials such as mesalime, electric arcfurnace dust and otation tailings for biopulp neutralisation have alsobeen proposed (Adamov et al., 2011; Gahan et al., 2010). Moreover,biooxidation has been used to increase the recovery of gold fromotation tailings. In the study by Kondrat'eva et al. (2012) biooxidationof otation tailings resulted in additional recovery of 2627% gold anda decreased cyanide consumption, compared to direct cyanide leaching.

Heap biooxidation treatment is generally consideredwhen the ore islow-grade, economics cannot sustain the cost of making a concentrate,the mineralogy is such that the refractory sulphides cannot be concen-trated, or the project is too small to support a high capital process(Brierley and Brierley, 2001). Heap biooxidation of refractory gold con-centrates has been conducted at the Agnes Gold Mine in South Africasince 2003 by the GEOCOAT process developed by GeoBiotics LLC(Ndlovu, 2008). In the process, sulphide concentrate slurry is coatedonto a crushed and sized carrier rock concentrate (Figs. 5 and 6). Thecoated material is stacked on an impervious pad for biooxidation(Ndlovu, 2008). After biooxidation, the heap is washed, the concentrateis separated from the carrier, washed and then the gold is dissolvedby cyanidation.

Biooxidation pretreatment of lower-value, refractory gold-bearingwhole ores can be conducted in heaps, similar to those used for second-

Au quarry mine USA Heap (whole ore) 10Agnes South Africa Heap (GEOCOAT)

References: 1) Brierley andBrierley, 2001, 2)Olson et al., 2003, 3) Rawlings, 2002, 4) Rawlin2008, 9) Harvey and Bath, 2007, 10) GeoBiotics, 2010, 11) Brierley, 2008, 12) Gericke et alary copper ores (Brierley, 2000). NewmontMining showed the practical-ity of heap biooxidation treatment of whole ores with a demonstration-scale heap at a 3.8-million t year1 facility at Gold Quarry Mine (Bhaktaand Arthur, 2002; Brierley, 2000). The ore is crushed to approximately

Fig. 5. GEOCOAT process for biooxidation of refractory gold concentrate at12.7 mm size (Brierley, 2000) and stacked on pads with an air-ventilation system at the base to supply oxygen and carbon dioxide tothemicroorganisms inoculated on to the ore (Olson et al., 2003). The ox-idation rate for the ore is typically between 0.30% and 0.34% sulphidesulphur oxidation/day. Therefore, an oxidation cycle of 100150 dayscould result in 3050% sulphidesulphur oxidation. Column test workon sulphide oxidation versus gold recovery indicated a diminishingreturn after 60% sulphide oxidation (Bhakta and Arthur, 2002). After100270 days of biooxidation, the ore is removed from the pretreatmentpads and cyanide leached in an oxide mill facility (Brierley, 2000; Olsonet al., 2003). Gold recovery ranges from 60% to 70% of the containedvalue with an ore grade range of 1.7 to 4.1 g t1 (Brierley, 2000). Goldcan be recovered from cyanide solutions using adsorption of gold ontoactivated carbon, which is then chemically stripped of gold. In a nalstep, gold is precipitated electrolytically or by chemical substitution(Reith et al., 2007b). More recently, non-cyanide lixiviants have beenevaluated as alternatives for leaching gold from biooxidised ores. Acolumn study simulating heap leaching indicated that thiosulphateleaching could result in similar recoveries as cyanide leaching frombiooxidised refractory gold ores (Gudkov et al., 2011).

Although not yet demonstrated in large-scale, in situ and in placeleaching methods could be attractive alternatives for low grade goldores that are too expensive to process using conventional open-pit orunderground mining and processing methods. With in situ leaching

2000present 2, 6, 720032006, 2009present 8, 9, 10

al., 2003, 5) doCarmo et al., 2001, 6) Bhakta andArthur, 2002, 7) Brierley, 2000, 8)Ndlovu,09, 13) Sovmen et al., 2009.the leach solution is injected into the subsurface ore body to extracttarget metals. The pregnant (metal-bearing) leach solution is collectedfrom production wells for metal recovery (Bosecker, 1997). In situleaching does not usually require extensive mine infrastructure and

the Agnes gold mine, South Africa. 2014 CSIRO. All Rights Reserved.

io

ld c

atink a

hi

edol

ins

Agn

74 A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083may reduce the visual impact of the mining operation. However, in situleaching does require relatively long contact times between oreminerals and uid andwell developed reservoir permeability. Accordingto Steven (2009) the porousmedium should have a hydraulic conductiv-ity of greater than 0.043 m d1 for successful in situ leach operation.Extensive knowledge of the hydrology and geology of the area and care-

b

Go

CorocH2SO4

Was

Oxidisto g

Microorganisms

PondNutrients

Water to treatment R

Make-up Water

Fig. 6. A simplied ow sheet of the GEOCOAT process at theful control of leaching solutions is required to prevent contamination ofnearby groundwater (Kinnunen, 2004; Nurmi, 2009). In place leachingis similar to in situ leaching but the ore body is fractured, for exampleby blasting, to improve the permeability before leaching (Waddenand Gallant, 1985). With a laboratory scale study, Kaksonen et al.(2014) showed that submerged oxidation of pyrite is possible using fer-ric iron that is biologically generated either externally or using under-ground aeration in the ore body. The simulated underground aerationand the presence of bioleaching microorganisms clearly enhanced theoxidation of pyrite. Moreover, microorganisms oxidised sulphur inter-mediates and thereby decreased the accumulation of elemental sulphur.The removal of pyrite and elemental sulphur is expected to enhance sub-sequent gold leachingwith chemical lixiviants and decrease the lixiviantconsumption (Kaksonen et al., 2014).

2.2. Microbial pre-treatment of refractory carbonaceous ores

Some gold ores have a carbon content that inhibits gold recoveryusing leaching or lixiviant processes and thus renders them refractory(Brierley and Kulpa, 1993). These include refractory carbonaceousand/or carbonaceous-sulphidic ores. The refractory carbon content is asignicant source of preg-robbing, which refers to its ability to removeor rob gold that has been leached out of the ore and held in pregnantlixiviant solution. It is believed that the carbonaceous component thatparticipates in the preg-robbing comprises an activated carbon-typematerial, long-chain hydrocarbons and organic acids, such as humicacid. Microbial pre-treatment processes have been developed to de-activate the carbonaceous component of these ores to prevent bindingof the dissolved gold onto this component. Brierly and Kulpa (1993)patented a treatment process where the ore is inoculated with amicrobial consortium of bacteria that comprises at least two species se-lected from the following: Pseudomonas (P.)maltophila, P. oryzihabitans,P. putida, P. uorescens, P. stutzeri, Achromobacter spp., Arthrobacter spp.,and Rhodococcus spp. The carbon-adsorbable blanking agent producedby the bacterial consortium is used together with a relatively highconcentration (25250 kg per ton of ore) of the chelating agent, ethyl-

Heap oxidation

oncentrate

g of support nd stacking

ng of support rock

concentrate d leaching

Support rock

returned to coating

ing of heap

Air

es gold mine, South Africa. 2014 CSIRO. All Rights Reserved.ene diamine tetraacetic acid (EDTA). The microbial deactivation ofcarboneous material can be conducted before, after or contemporane-ouslywith biooxidation of sulphidicminerals. Yen et al. (2009) patentedan alternative process that is based on the use of fungal agents and/orculture media. Although any suitable fungi may be employed, thepreferred heterotrophic agents listed in the patent are white rot fungi,such as Trametes spp., Phanerochaete spp., Phlebia spp., Cyathus spp.,and Tyromyces spp. It is belived that some carbonaceous materials areconverted into carbon dioxide by some fungiwhile other fungi passivatethe preg-robbing capacity of carbonaceous materials (Yen et al., 2009).

2.3. Permeability enhancement

One of the critical factors for the success of low-grade gold ore in situand in place leaching is the permeability of the ore body. Microorgan-isms can contribute to the breakdown of rock forming minerals byboth biochemical mechanisms and physical (mechanical) forces suchas through the action of microscopic fungi that spread within cracksand even through entire mineral bodies (Brehm et al., 2005; Jongmanset al., 1997). Some microorganisms promote rock weathering bymobilising mineral constituents with the inorganic and organic acidsor ligands that they excrete. Others promote rock weathering byredox attack of mineral constituents such as Fe and Mn (Ehrlich,1998). Biochemical breakdown of rock forming minerals can result inmicrotopographic change of mineral surfaces producing: pitting andetching of their surfaces, mineral displacement reactions, widening ofpores andmineral interphases, and even complete dissolution ofmineralgrains (Brehmet al., 2005; Burford et al., 2003; Ehrlich, 1998; Kumar andKumar, 1999). Biological solutions have also been reported to permeatemicrofractures and affect the wettability of ores and solution contact

75A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083with leachable gold due to surfactant properties (Thompson andMacCulloch, 2004).

2.3.1. Removal of passivating S0- and Fe3+-layersThe oxidation of sulphide minerals is thought to be hindered by the

formation of passivating layers of elemental sulphur, polysulphides andjarosite (Stott et al., 2000). Bioleaching can be enhanced by usingmicroorganisms capable of removing passivating layers from mineralsurfaces. By doing this microorganisms may also be able to enhancethe permeability of ore deposits. For example some acidophilicmicroor-ganisms, such as At. ferrooxidans are capable of oxidising sulphur underanaerobic conditions using ferric iron as the terminal electron acceptor(Pronk et al., 1992):

S0 6Fe3 4H2O SO42 6Fe2 8H 8

The generation of acid in this process decreases pH, which in turndecreases the precipitation of ferric iron as passivating iron hydroxycompounds (Nurmi, 2009). In addition to At. ferrooxidans, severalother acidophilic microorganisms can also reduce ferric iron, includingAt. ferrivorans, Acidiferrobacter thiooxydans, Ferrimicrobium acidiphilum,Acidimicrobium ferrooxidans, Ferrithrix thermotolerans, several Acidiphilumspp., Acidocella spp., Acidobacterium spp., Alicyclobacillus spp., Sulfobacillusspp., Acidiplasma spp. and Ferroplasma spp. (Johnson et al., 2012). Ferriciron reducing microorganisms can catalyse reductive dissolution of ironhydroxy compounds which contain ferric iron, such as jarosite, goethite,or schwertmannite (Johnson and Hallberg, 2009). Reaction 9 showsthe reductive dissolution of schwertmannitewith glucose as an electrondonor (Coupland and Johnson, 2008):

3Fe8O8 OH 6 SO4 C6H12O6 6H2O 24Fe2 6CO2 3SO24 42OH 9

Reductive dissolution of iron hydroxy compoundswould require theaddition of a suitable electron donor for the microorganisms. Althoughthe addition of organic substrates into the subsurface may enhancethe reductive dissolution of iron hydroxy compounds, the organicsubstrate may also be utilised by microorganisms, such as sulphatereducers, which can decrease the solubility of gold.

2.3.2. Dissolution of silicate mineralsSomemicroorganisms, such as bacteria and fungi are able to acceler-

ate dissolution of silicates and aluminosilicates. According to Ehrlich(1996) their action on these minerals has been characterised as non-enzymatic. The dissolution mechanisms may involve production ofmetabolic products such as organic acids that act as acidulants and/or li-gands, alkalinity in the form of NH3, and capsular slime (acid polysac-charide) from bacteria (Ehrlich, 1996). Among the acids, 2-ketogluconic acid formed by some bacteria, and citric and oxalic acidsformed by some fungi, have been reported to be effective in the dissolu-tion of silicates (Duff et al., 1963; Ehrlich, 1996; Vandevivere et al.,1994). The organic acids furnish protons that help in breaking SiOand AlO bonds through protonation. Some of the acids may also actas ligands that pull cations from the framework of the crystal lattice, fa-cilitating subsequent breakage of framework bonds. Some bacterialslimes (acid polysaccharide) have been reported to form complexeswith silicate leading to silicate dissolution (e.g. Ehrlich, 1996; Liu et al.,2006; Malinovskaya et al., 1990).

Quartz comprises 20% of the volume of the exposed Earth's crust andis one of themost resistant of rock formingminerals (Brehmet al., 2005;White and Brantley, 1995). Its rate of dissolution is slow, approximately1017 mol cm1 s1 at 40 C at near neutral pH in purewater, becausethe activity required to break SiO bonds is high (Brehm et al., 2005).As a component of rocks (e.g. granite, gneiss, sandstone), quartz crystals

and grains have a higher resistance to weathering processes than manyother commonminerals such as feldspar and mica (Brehm et al., 2005).At pHvalues lower than 3.5, quartz dissolution isminimal, althoughme-chanical processes of grain diminution may still continue (Brehm et al.,2005). Quartz solubility increases signicantly in alkaline conditions ofpH 9 and above (Brehm et al., 2005). A number of biological processescan generate alkalinity or consume acidity and therefore have potentialfor increasing pH and thus quartz dissolution. These include: photosyn-thesis (Brehm et al., 2005; Johnson, 2000; Robb and Robinson, 1995;van Hille et al., 1999), denitrication (Johnson, 1995; Kalin et al.,1991), hydrolysis of urea (Fujita et al., 2000), ammonication,methanogenesis, and reduction of iron and sulphate (Johnson, 1995,2000; Kalin et al., 1991; White et al., 1997) (Table 2).

Brehm et al. (2005) reported that natural biolms composed ofdiatoms, heterotrophic bacteria and cyanobacteria can actively attackquartz and glass. Microscopic analysis of the quartz crystal from atepui, a type of quartzitic tabular mountain found in the Guiana High-lands of South America, revealed that the associated biolms can createa local shift in the pH from 3.4 (pH of the water on the tepui), to N9(necessary for quartz dissolution) (Brehm et al., 2005). The quartzcovered with biolm was partially perforated to a depth of more than4 mm (Brehm et al., 2005). However, Brehm et al. (2005) estimatedthat the resident microbial community had been affecting the mineralsurface considerably longer than 10 years.

A consortium of diatoms (eukaryotic algae) and heterotrophicbacteria created depressions or pitted zones to window glass in a9 months study (Brehm et al., 2005). The diatoms and their accompa-nying bacteria were embedded in large amounts of extracellular poly-saccharides covering the surface of the glass. Brehm et al. (2005)suggested that diatoms produce polysaccharides useful for bacterialmetabolism and survival, whereas bacteria with their leaching activityprovide silicon ions for diatom frustule construction. The use of photo-synthetic diatoms and cyanobacteria would not be applicable in darksubsurface in situ leaching environments due to the lack of sunlight.

Quartz often contains iron as an impurity (tyriakov et al., 2003).tyriakov et al. (2003) studied the biodestruction and deferrisation ofquartz sands with Bacillus spp. The bioleaching experiments showedthat Bacillus spp. can solubilise iron, silica, and aluminium from quartzsands and reduce the iron oxyhydroxides present as impurities.

3. Gold solubilisation through biooxidation and complexation

A number of chemical and biologically produced lixiviants havebeen assessed for their ability to oxidise and complex gold as alterna-tives to chemical cyanide solubilisation.

3.1. Thiosulphate

One of the most potential alternatives to cyanide systems is theleaching of gold with thiosulphate in the presence of co-ligands, suchas ammonia and oxidants such as Cu2+ (Aylmore and Muir, 2001;Reith et al., 2007b; Wan and LeVier, 2003). The leaching reaction isdescribed as follows (Reith et al., 2007b):

Au 5S2O32 Cu NH3 42 Au S2O3 23h i

Cu S2O3 35 4NH3 10

Au(I)thiosulphate complex is stable inmildly acidic to highly alkaline pH(510), and moderately oxidising to reducing conditions (Eh 0.170.76 V) (Reith, 2003).

Thiosulphate heap leaching has already been used in the CarlinNevada operation of Newmont Mining. A combination of biooxidationand chemical thiosulphate heap leaching is applied for carbonaceous/high sulphide ores and a direct thiosulphate heap leaching process forcarbonaceous/low sulphide ores (Wan and LeVier, 2003). Until 2003 a

total of 1.24 million tonnes of low-grade refractory gold ores were

OH

m et

76 A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083successfully processed with ammonium thiosulphate at the Nevadaoperation (Wan and LeVier, 2003).

Thiosulphate and ammoniumare produced and excreted by bacteriaand actinomycetes during a number of metabolic reactions (Reith et al.,2007b). Ammonium is commonly produced through the hydrolysis ofurea by a wide range of yeasts and bacteria, including many alkaliphilicBacillus spp. (Reith et al., 2007b; Schmidt Mumm and Reith, 2007).Sulphate-reducing bacteria (SRB), which are common in anoxicsulphate-containing soils, form thiosulphate under certainenvironmental conditions, such as during reduction of sulphite withH2 and formate (Fitz and Cypionka, 1990; Reith et al., 2007a, 2007b).A common soil actinomycete, Streptomycetes fradiae, producesthiosulphate when metabolising sulphur from cystine (Kunert andStransky, 1988; Reith et al., 2007a). Thiosulphate has also beensuggested to be the main intermediate product of the biooxidationof acid-insoluble sulphides, such as pyrite (FeS2) and molybdenite(MoS2) and is oxidised further to sulphate (Schippers and Sand, 1999):

FeS2 6Fe3 3H2O S2O32 7Fe2 6H 11

S2O32 8Fe3 5H2O2SO42 8Fe2 10H 12

Gold solubilisation via the thiosulphate mechanism is expected inorganic carbon matter-poor environments, for example in primary sul-phide bearing deposits (Reith et al., 2007a). Reith and McPhail (2006)studied the solubilisation of sub-microscopic gold in carbon-limitedquartz vein materials with arsenopyrite and pyrite from Tomakin ParkGold Mine in New SouthWales, Australia. Reith et al. (2007a) proposedthat the gold solubilisation was mediated by microbially producedthiosulphate. A maximum of 550 ng gold per gram (dry weight quartzvein material, particle size b200 m) was solubilised in a biologicallyactive agitated slurry after 35 days of incubation and the concentra-tion decreased thereafter. In contrast, a sterile control systemshowed a ten times lower concentration of solubilised gold (Reithand McPhail, 2006; Reith et al., 2007a). The gold grade of the quartzvein material was not reported, and hence % gold extraction cannot be

Table 2Alkalinity producing microbially catalysed bioprocesses.

Process Reaction

Photosynthesis H2O + CO2 + light O2 + CH2OAmmonication Organic-N NH3Urea hydrolysis CO(NH2)2 + 2H2O 2NH4+ + CO32

Methanogenesis CH3COO + H2O CH4 + HCO3

Sulphate reduction SO42 + 2CH2O H2S + 2HCO3

Denitrication 6NO3 + 5CH3OH 5CO2 + 3N2 + 7H2O + 6OH

Ferric iron reduction withorganic electron donors

4Fe(OH)3 + CH2O 4Fe2+ + H2CO3 + 2H2O + 8

References: 1) Robb and Robinson, 1995; 2) van Hille et al., 1999; 3) Johnson, 2000; 4) Brehcalculated.

3.2. Organic acids

Organic acids (e.g. humic and fulvic acids, amino acids and carboxyl-ic acids) have been shown to promote the solubilisation of native gold insome experiments, whereas in other studies under different conditionsnative goldwas not oxidised and the formation of gold colloidswas pro-moted (Baker, 1978; Fetzer, 1934, 1946; Reith et al., 2007a; Wood,1996). A number of studies have indicated that amino acids producedby heterotrophic microorganisms, such as Bacillus (B.) subtilis, B. alvei,B. megaterium, B. mesentericus, Serratia marcescens, P. uorescens andP. liquefaciens, can enhance gold solubilisation by forming gold-aminoacid complexes (Korobushkina et al., 1974; Reith et al., 2007a). Accord-ing to Korobushkina et al. (1974), aspartic acid, histidine, serine, alanineand glycine played a substantial part in gold dissolution by cultures iso-lated from gold-bearing deposits. Amino acid production by the strainswas increased bymutagenic factors (ultraviolet rays and ethylenimine)and the solubility of gold increased in the presence of an oxidising agent(2 g L1 sodiumperoxide) under alkaline conditions (pH 910). Disso-lution of gold by puried amino acid fractions yielded solutions with upto 1415 mg L1 of gold in 20 days. The maximum concentration was35 mg L1 with no more than 0.20.3 mg L1 in the control experi-ment. The stability of gold-amino acid complexes varies with theirredox potentials. The complex forming capacity of amino acids may beranked according to the redox potentials as follows: cysteine N -histidine N asparagines N methionine N glycine, alanine, valine, phe-nylalanine (Korobushkina et al., 1983). Jingrong et al. (1992)suggested that the nitrogen atom in the amino group (NH2) shows astrong tendency of complexing gold, and the oxygen atom in the car-boxyl group (COO) also contributes to complexation. Jingron et al.(1992) also reported that the solubilisation of gold by amino acids de-pends on pH and temperature. Salt- and alkaline-soluble proteins andalso water- and alcohol-soluble proteins were less effective in dis-solving gold with soluble concentrations reaching 2.23.3 mg L1

and 0.150.57 mg L1, respectively in 20 days at pH 910(Korobushkina et al., 1983).

In the Tomakin Park GoldMine studywith gold-containing soils richin organic matter, biologically active samples displayed up to 80 wt.%gold solubilisation (original gold concentration 1453 ng g1 d.w. soil)within 45 days of incubation under aerobic conditions, after whichgold was re-adsorbed to the solid soil fractions (Reith and McPhail,2006). In the early stages of incubation themicrobial community appar-ently produced an excess of amino acids (up to 64.2 M of free aminoacids measured within the rst 20 days of incubation), which formedcomplexes with gold. However, in the later stages of the incubationthe microbial community metabolised these gold-complexing ligands(free amino acid concentration decreased to around 8 M by day50), and gold, which apparently became unstable in the solution,was re-adsorbed to the solid soil fractions. During the experimentthe bacterial community structure changed from a carbohydrate-and polymer-utilising community to a carboxylic- and amino-acid

Comments Reference(s)

Requires sunlight would not work underground 1, 2, 3, 4Requires organic N 3, 5, 6, 7Requires aerobic conditions and an organic substrate 8Requires anaerobic conditions 3, 5, 6, 7Requires anaerobic conditions, sulphate,and an electron donor

3, 5, 6, 7

Requires anaerobic conditions, nitrate or nitrite,and an electron donor

5, 6

Requires anaerobic conditions, Fe3+ compounds,and an electron donor

3, 5, 6, 7

al., 2005; 5) Kalin et al., 1991; 6) Johnson, 1995; 7)White et al., 1997; 8) Fujita et al., 2000.utilising community concurrently with the change from goldsolubilisation to re-adsorption. The microbiota of gold-containingsoils was also capable of dissolving gold from added gold pellets,resulting in concentrations higher than the original soil sample andthe presence of biolms on the surfaces of the gold pellets. In con-trast, the microbiota from soils 100 m from the mineralisation,which displayed only background gold values, did not mobilisegold nor form biolms on added gold pellets indicating that the mi-crobiota were different or acted differently in gold-containing soils(Reith and McPhail, 2006).

The interaction of gold and organic matter involves mostly electrondonor elements, such as nitrogen, oxygen and sulphur, rather thancarbon (Reith et al., 2007a). Vlassopoulos et al. (1990) showed thatgold binds preferentially to organic sulphur under reducing conditions,and that complexation with organic nitrogen and carbon is more

neutral pH, cyanide mainly occurs as volatile HCN because of its pKavalue of 9.3. However, in the presence of salts and metal ions, volatility

77A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083is reduced, and thus biologically produced cyanide may directly affectgold solubilisation (Faramarzi and Brandl, 2006; Reith et al., 2007a).With cyanide Au+ forms a strong dicyanoaurate complex which is sta-ble over a wide range of redox and pH conditions (Reith et al., 2007a):

4Au 8CN O2 2H2O4AuCN2 4OH 13

An in vitro study using the cyanogenic bacterium Chromobacterium(Chr.) violaceum showed that biolms grown on gold-covered glassslideswere able to solubilise 100% of the gold within 17 days, with con-centrations of gold and free cyanide in solution reaching 35 and14.4 mg L1, respectively (Campbell et al., 2001). When incubatingChr. violaceum with biooxidised gold concentrate up to 0.34 mg L1

gold and 9 mg L1 CN was detected in solution within 10 days(Campbell et al., 2001). In a more recent study Fairbrother et al.(2009) examined the effect of cyanide production by Chr. violaceumon ultra-at gold foil by incubating the bacteria and foil in peptonemeat extract for up to 56 days. Total concentrations of solubilised goldincreased throughout the experiment and after 56 days 74.3 g L1 ofgold was detected in solution and 51.3 g L1 reversibly or irreversiblyboundwith cells. Fairbrother et al. (2009) noted that the lower concen-trations when compared to those observed by Campbell et al. (2001)may have been due to the low surface roughness of the ultra at goldfoil used in the more recent study. Shin et al. (2013) suggested oregrinding and pre-growing Chr. violaceum for increasing gold recovery.A patent has already been granted for the biohydrometallurgicalimportant in oxidising environments. Gold solubilisation via complexa-tion by organic acids may occur in organic matter-rich top and rhizo-sphere soils, where plant exudates may directly lead to goldsolubilisation or provide nutrients for organic acid-excreting microor-ganisms (Reith et al., 2007a).

Some amino acids are also precursors for the microbial production ofother gold-complexing ligands, e.g. cystine is a precursor for thiosulphate(Kunert and Stransky, 1988; Reith et al., 2007b) and glycine is a precursorfor cyanide (Fairbrother et al., 2009; Reith et al., 2007a, 2007b; Rodgersand Knowles, 1978). Moreover, amino acids have been shown tocomplex Cu2+ during thiosulphate leaching of gold-bearing pyrite. Thisdecreased thiosulphate consumption due to reduced interaction be-tween thiosulphate and the copper complexes (Feng and van Deventer,2011). As with biogenic thiosulphate production, the transient stabilityof the amino acids may indicate that economic exploitation of aminoacids for gold solubilisation may be challenging.

Current industrial amino acid production is a multi-billion dollarbusiness, with annual production estimated to be millions of tons.Amino acids are used in a number of applications such as food additives,pharmaceuticals, cosmetics, polymer materials, biofuels and antibiotics(Park and Lee, 2008). The development of efcient amino acid produc-ing strains has traditionally involved multiple rounds of random muta-tion and selection. More recently approaches for strain developmenthave shifted to targeted engineering strategies which purposefullymodify genes and pathways towards enhanced production of desiredamino-acids (Park and Lee, 2008).

3.3. Biogenic cyanide

Many soil bacteria (such as P. uorescens, P. aeruginosa, P. putida,P. syringae and B.megaterium), fungi and plants can produce and excretecyanide (Reith et al., 2007a). Cyanide has no apparent function inprimary metabolism, is optimally produced during growth limitationand may offer the producer, which is usually cyanide tolerant, aselective advantage by inducing cyanide toxicity in other organisms(Bakker and Schippers, 1987; Castric, 1975; Reith et al., 2007a). Atprocessing of gold-containing ores using cyanide producingmicroorganisms, such as Chr. violaceum and Chlorella (Chl.) vulgaris(Kleid et al., 1995; Krebs et al., 1997).

Cyanide producing microorganisms have also been applied to leachgold from metal-containing waste materials (Brandl et al., 2008;Faramarzi et al., 2004). P. plecoglossicida solubilised gold from shreddedprinted circuit boards producing up to 500 mg L1 [Au(CN)2] in 80 h,corresponding to gold concentration of 442 mg L1 and a 69% dissolu-tion of the gold added (Faramarzi and Brandl, 2006; Reith et al., 2007a).Faramarzi et al. (2004) and Brandl et al. (2008) demonstrated goldsolubilisation from shredded printed circuit boards with Chr. violaceumand measured dicyanoaurate concentrations corresponding to 14.9%and 68.5% gold dissolution, respectively in 7 days, after a 3-day lagphase. A two-step bioleaching process has been proposed to overcomethe toxic effects of electronic waste on bioleaching cultures (Brandlet al., 2001; Mishra and Rhee, 2010; Pradhan and Kumar, 2012).Pradhan and Kumar (2012) applied a two-step bioleaching processto rst generate cyanide forming biomass in the absence ofelectronic waste followed by the addition of electronic waste formetal solubilisation. Chr. violaceum was capable of leaching 69% ofgold and mixture of Chr. violaceum and P. aeruginosa exhibited 73%gold leaching at an electronic waste concentration of 1% w/v (Pradhanand Kumar, 2012). When P. uorescence was applied for electronicwaste leaching, dicyanoaurate did not remain stable in solution withprolonged incubation times, probably due to sorption onto biomass orbiodegradation because cyanides can serve as carbon or nitrogen source(Brandl et al., 2008). Kita et al. (2006) showed that increased dissolvedoxygen concentration enhanced gold solubilisation by Chr. violaceumfrom electronic waste whereas Chi et al. (2011) showed that increasingpH from 8 to 11 increased gold leaching by the same species. Pham andTing (2009) reported that biooxidation pretreatment of electronicwaste with At. ferrooxidans removed most of the copper present in thewaste and signicantly increased the gold recovery in subsequentleaching with Chr. violaceum.

Amino acids, such as glycine, can function as metabolic precursorsfor microbial cyanide production (Fairbrother et al., 2009; Reith et al.,2007a; Rodgers and Knowles, 1978). The highest concentration of freeglycine in solution in the Tomakin ParkGoldMine soil studywas detectedafter 20 days of incubation (Reith and McPhail, 2006), and a cyanideconcentration up to 0.36 mg L1 (soil solution) was measured inanother Tomakin soil study suggesting that the solubilisation of gold,as a dicyanoaurate complex,may occur in addition to gold solubilisationwith amino acids (Reith et al., 2007a).

Gold solubilisation via cyanide mechanisms may occur in rhizo-sphere soils with tops rich in organic matter, where plant exudatesmay lead directly to gold solubilisation or provide nutrients for cyanideexcreting microorganisms (Bakker and Schippers, 1987; Reith et al.,2007a). Microbial in situ cyanide generation has also been suggestedas away to reduce cyanide transport and reduce the total quantity of cy-anide required (Zammit et al., 2012). Themajor challengewithmicrobi-al generation of cyanide is the rate of production and cost (Zammit et al.,2012). Industrial application of this process would require that itbe more cost effective than chemical production and use of cyanide,forwhich an industrial base is already available.Moreover, using biolog-ically produced cyanide for in situ leaching may be prohibitive dueto the environmental issues. It would likely to be more acceptable toproduce cyanide in bioreactors and then use the biogenic cyanide intraditional processing methods, such as reactors or heaps, as suggestedby Zammit et al. (2011).

Pintain Systems Inc. demonstrated enhanced gold recovery duringbiological detoxication of cyanide in heap leached spent ore at CrippleCreek and Victor Mining Company Ironclad Mine near Victor, Colorado,USA. Gold recovery was at least two times higher than that expectedfrom a normal heap rinse operation producing an additional 156 kg ofgold from ve million tons of spent ore (Beckman and Thompson,2004; Thompson and MacCulloch, 2004; Thompson et al., 1998).

The mechanism of the biologically enhanced gold solubilisation was

not disclosed. According to Thompson and MacCulloch (2004) Pintailhas developed a culture collection of bacteria and fungi which havedemonstrated a variety of gold solubilisation mechanisms. With acolumn study they showed how various microbial strains were able tosequentially recover gold from ore originating from Buffalo Valley pitnear Battle Mountain, Nevada, USA. Beckman and Thompson (2004)used the term BioLix for biologically-derived lixiviants for preciousmetals (gold and silver) recovery. The BioLix process is claimed to bebased on the generation of organic lixiviants with non-pathogenic nat-urally occurring microorganisms. For BioLix applications micro-organisms are adapted to the target ore and cultured in a proprietarybroth to increase cell numbers. Thereafter, a proprietary enzymaticinducing agent is added to initiate the microbial production of thelixiviants. The microbial solution is then applied to the ore for thesolubilisation and complexation of precious metals (Beckman andThompson, 2004).

3.4. Iodide

One promising inorganic lixiviant for gold is the iodide(I)iodine(I2) system. The most likely electrochemical half-reactionsinvolved in the dissolution of gold are the following (Davis and Tran,1991):

I2 H2OH I HIO 19

I2 I I3 20

These equilibria are inuenced by temperature, pH, and concentrationof I and I2 in the solution (Davis and Tran, 1991).

Major pathways in the biogeochemical cycling of iodine are oxida-tion and reduction of inorganic iodine species, the volatilisation oforganic iodine compounds into the atmosphere, accumulation of iodinein living organisms, and sorption of iodine by soil and sediments (Fig. 7).Considerable geochemical evidence has indicated that these processesare inuenced or controlled bymicrobial activities, although the precisemechanisms involved are still unclear (Amachi, 2008). Thus microor-ganismsmay help to regenerate the iodideiodine lixiviant by oxidisingI to I2, and on the other hand, contribute to the loss of the lixiviantthrough volatilisation, accumulation and sorption. Some microorgan-isms can also reduce iodate (IO3) to I (for a review, see Amachi, 2008).

Microbial IO3 reduction to I is still poorly understood largely dueto the limited number of isolates available aswell as the paucity of infor-mation about key enzymes involved in the reaction. The average totalconcentration of dissolved iodine in seawater is 0.45 M, and the pre-

HIO

I-(

78 A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083Anodic : Au 2I AuI2 e 14

Cathodic : I3 2e 3I 15

Yielding the overall reaction:

2Au II3 2AuI2 16

Gold dissolution may also occur according to the following overallreaction (Angelidis et al., 1993):

2Au 3I3 2AuI4 I 17

As with other halogens, the aqueous iodideiodine system consistsof several known species, I2, I, I3, IO and HIO existing in equilibrium(Davis and Tran, 1991):

HIOH IO 18

I2 (0)

Abioticoxidation

CH2I2CH2ClI

Microbial oxidation and volatilisation

SorptionFig. 7. Contribution of bacteria (solid arrows) to the biogeochemical cycling ofdominant chemical forms are I and IO3. Thermodynamically, the con-centration ratio between IO3 and I in oxygenated seawater at pH 8.1and pE 12.5 should be 3.2 1013, indicating that IO3 is the more stableform. However, in deep waters (Nakayama et al., 1989), anoxic basins(Farrenkopf et al., 1997; Wong and Brewer, 1977) and pore waters ofmarine sediments (Muramatsu et al., 2007) I is often highly enrichedat concentrations of several M to more than one mM (Amachi, 2008).In addition to abiotic chemical reduction of IO3 and microbialremineralisation of organic iodine compounds, microbial reduction ofIO3 is likely to be an important process to maintain reduced form ofiodine in these environments (Amachi, 2008).

Recently, an IO3-reducing Pseudomonas sp. strain SCT was isolatedfrom marine sediment slurry by Amachi et al. (2007a). The strainreduced 200 M IO3 to Iwithin 12 h in an anaerobic culture contain-ing 10 mMnitrate, but could not growwith 5 or 10 mM IO3 as the soleelectron acceptor. However, it showed signicant growth when muchlower concentrations (2, 3, and 4 mM) of IO3 were added as theelectron acceptor. The growth was nearly proportional to the IO3

concentration in the medium. The strain used malate, glycerol, lactate,

(+1)

IO3-(+5)

-1)

CH3I

Microbial reduction

Microbial volatilisation

Accumulation and sorption

Abioticoxidation iodine (modied from Amachi, 2008). 2014 CSIRO. All Rights Reserved.

which accumulates gold in an EPS capsule. Gold was sequestered bycultures whichwere able to form the capsule whereasmutants withoutcapsules were not able to do so (Quintero et al., 2001). Accumulation ofgold into the EPS also contributed to the higher viability of P. aeruginosasubjected to 0.1 mMAu3+ chloridewhen grown as a biolm comparedto free planktonic cells (Karthikeyan and Beveridge, 2002). Kenney et al.(2012) used non-metabolising cells of B. subtilis and P. putida andachieved over 85% gold removal from a solution with initial gold con-centration of 5 ppm at pH b 5 in 2 h. At increasing pH the adsorptionslowed down. Dementyev and Voiloshnikov (2011) reported adsorp-tion activity of A. niger and A. orizae for dissolved gold to be as high asthat of industrial activated carbon, and for gold colloids it was 810times higher than for activated carbon. In pilot plant test the goldrecovery with the fungal biomass was 9698%.

4.2. Enzymatic reductive precipitation

Some bacteria and archaea are able to precipitate gold by reducingAu3+ to Au0 (He et al., 2007; Kashe et al., 2001). Some species, includ-ing Pyrobaculum islandicum, Pyrococcus furiosus, Shewanella algae andD. vulgaris precipitate Au0 extracellularly, whereas others, such asGeobacter ferrireducens, precipitate Au0 within the periplasmic space.According to Kashe et al. (2001) the Au3+ reduction in dissimilatory

3+

79A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083succinate, acetate, and citrate as electron donors. The strain did notreduce IO3 under aerobic conditions (Amachi et al., 2007a).

Direct microbial reduction of IO3 has also been demonstratedwith anaerobic cell suspensions of the sulphate-reducing bacteriumDesulfovibrio (D.) desulfuricans and the dissimilatory Fe3+-reducingbacterium Shewanella (S.) putrefaciens which were able to reduce IO3

at pH 7 in 10 mM 4-2-hydroxyethyl-1-piperazineethanesulphonicacid (HEPES) buffer (Councell et al., 1997). D. desulfuricans was alsoable to reduce 96% of an initial 100 M IO3 to I at pH 7 in 30 mMNaHCO3 buffer, whereas S. putrefaciens was not. Both soluble ferrousiron and sulphide, as well as iron monosulphide (FeS) were shown toabiologically reduce IO3 to I . The study indicated that ferric ironand/or sulphate-reducing bacteria are capable of mediating direct,enzymatic, as well as abiotic reduction of IO3 in natural anaerobicenvironments (Councell et al., 1997).

The oxidation of I to IO3 does not occur spontaneously in slightlyalkaline solutions like seawater, since the rst step in the process,i.e. the oxidation of I to I2 is thermodynamically unfavourable at thepH of seawater. However, once I2 is formed, the hydrolysis of I2 to formHIO will occur rapidly. Moreover, HIO disproportionates spontaneouslyto form IO3 (Amachi, 2008). A number of bacteria have been shown tooxidise I. In 1968 Gozlan reported the isolation of an I-oxidising bac-terium from experimental seawater aquaria (Gozlan, 1968). The isolate,later named as Pseudomonas iodooxidans, was a heterotrophic Gram-negative bacterium which oxidised I to I2 through an extracellularperoxidise with hydrogen peroxide as an electron acceptor (Gozlanand Margalith, 1973, 1974):

H2O2 2I 2HI2 2H2O 21

More recently, Fuse et al. (2003) and Amachi et al. (2005b) iso-lated I-oxidising bacteria from marine environmental samples.The bacteria were afliated with the -subclass of Proteobacteria.Some of the strains were most closely related to Roseovarius tolerans(9498% 16S rRNA gene sequence similarity) and others were relatedto Rhodothalassium salexigens (8991% 16S rRNA gene sequencesimilarity). The I-oxidising reaction was mediated by an extracellularoxidase that requires oxygen (Amachi et al., 2005b):

4I O2 4H2I2 2H2O 22

Although the oxidation of I by oxygen as an electron acceptor is en-ergetically favourable (G0 = 56 kJ per reaction), the extracellularnature of the enzyme implies that energy conservation by this reactionis not possible (Amachi et al., 2005b). I-oxidising bacteria seem toprefer I-rich environments (Amachi et al., 2005b). I may enhancethe competitive advantage of I-oxidising bacteria over competingmicroorganisms by toxic iodine species (Amachi, 2008). I2 producedby I-oxidising bacteria is a highly active oxidising agent, and has strongbactericidal, fungicidal, and sporicidal activities (McDonnell and Russell,1999).

Microbially mediated cycling of iodine to regenerate the iodideiodine lixiviantmay hold potential for gold leaching. However, the prac-tical feasibility of the concept has not been demonstrated. On the otherhand microbial volatilisation, accumulation and sorption of iodine leadto the loss of the lixiviant. All of the processes occur at around neutralpH and are stimulated by the supplementation of organic compounds.The IO3 reduction is favoured by anoxic conditions whereas all theother biologically catalysed processes are favoured by oxic conditions(Table 3).

4. Gold recovery and/or loss through bioprocesses decreasinggold solubility

In contrast to most other metals, gold is extremely rare, inert,

and unstable as a free ion in aqueous solutions under atmosphericconditions (Reith et al., 2007a). Gold complexes can be highly toxic tomicroorganisms. Hence, microorganisms have many mechanismsto deal with toxic gold complexes and are able to precipitate goldintra- and extracellularly, and in products of their metabolism, such asexopolysaccharide (EPS) and sulphide minerals (Reith et al., 2007a).Some of these mechanismsmay hold potential for gold recovery, othersmay result in unwanted gold loss from pregnant leach solutions.

4.1. Biosorption and accumulation of gold

Many bacteria (e.g. P. maltophilia, B. subtilis, Escherichia coli), actino-mycetes (e.g. Streptomyces albus, S. fradiae, Saccharopolyspora erythraea),algae (e.g. Ascophyllum nodosum, Chl. vulgaris, Sargassum natans), yeast(Candida utilis, Saccharomyces cervisiae) and fungi (e.g. Aspergillus(A.) niger, Cladosporium cladosporioides, Fusarium oxysporum, Rhizopusarrhizus) can contribute to passive sorption of gold from solution (Cuiand Zhang, 2008; Labeda, 1987; Reith et al., 2007a). Somegroups of bac-teria have anunusual capacity to sequester gold andbioconcentrate it tovery high levels. One such strain is Hyphomonas adhaerens MHS-3,

Table 3Examples of bacteria participating in geochemical cycling of iodine. All of the listedbacteria grow at near neutral pH and benet from supplementation with organiccompounds.

Process Microorganisms involved Optimalconditions(oxic/anoxic)

Reference(s)

Reductionof IO3 to I

Denitrifying bacteria:e.g. Pseudomonas sp. strain SCT;sulphate-reducing bacteriae.g. Desulfovibrio desulfuricans;Fe3+ reducing bacteria:e.g. Shewanella putrefaciens

Anoxic 1, 2

Oxidationof I to I2

Pseudomonas iodooxidans,-subclass of Proteobacteria,e.g. strains related to Roseovarius sp.and Rhodothalassium sp.

Oxic 3, 4, 5, 6

Volatilisation Variovorax sp. Oxic 7Rhizobium sp.

Accumulation Arenibacter sp. Oxic 8Sorption Not reported Oxic 6, 9, 10

References: 1) Amachi et al., 2007a; 2) Councell et al., 1997; 3) Gozlan and Margalith,1974; 4) Fuse et al., 2003; 5) Amachi et al., 2005b; 6) Amachi, 2008; 7) Amachi et al.,2001; 8) Amachi et al., 2005a; 9) Koch et al., 1989; 10) Bird and Schwartz, 1996.Fe reducers appears to be an enzymatically catalysed reaction

80 A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083which is dependent upon the presence of a specic electron donor,hydrogen, since alternative electron donors, such as lactate did not pro-mote Au3+ reduction. The Fe3+-reducingmicroorganisms that reducedAu3+ did not appear to have a signicant capacity for adsorptionof Au3+ prior to reduction, because there was no loss of Au3+ fromsolution in the absence of hydrogen or when cells were incubated attemperatures that inhibited metabolism. This suggests that the Fe3+-reducing microorganisms reduced Au3+ prior to, or simultaneouslywith, the adsorption of gold onto the cell surface (Kashe et al., 2001).This mechanism appears to be clearly different from the mechanism ofaerobic microorganisms which adsorb Au3+ on the cell surface, withthe subsequent reduction of the adsorbed Au3+ to Au0, although themechanisms of these interactions are also poorly understood (Kasheet al., 2001; Savvaidis et al., 1998).

Enzymatically catalysed precipitation of gold has been suggested tohave led to the formation of gold-bearing biomineralisations in variousenvironments (Fairbrother et al., 2013; Reith et al., 2006; Reith et al.,2007a). Fairbrother et al. (2013) used quartz sand packed columns toassess the biomineralisation potential of Cupriavidus metallidurans,which can reductively precipitate gold as nanoparticles (Reith et al.,2009). While abiotic control columns retained only b30 wt.% of thegold added as Au(I)-thiosulphate, the inoculated columns achievedN99 wt.% gold removal. The synthesis of gold nanoparticles has alsobeen successfully demonstratedwith a variety of other microorganisms(Gwynne, 2013) including bacteria, such as E. coli (Du et al., 2007),Marinobacter pelagius (Sharma et al., 2012) and Delftia acidovorans(Johnston et al., 2013), fungi, such as Verticillium luteoalbum (Gerickeand Pinches, 2006), yeast, such as Yarrowia lipolytica (Agnihotri et al.,2009), and actinomycete, such as Rhodococcus (Ahmad et al., 2003a)and Thermonospora (Ahmad et al., 2003b). Both intracellular andextracellular formation of nanoparticles has been reported (Gerickeand Pinches, 2006; Johnston et al., 2013). Gold nanoparticles can beused in several applications such as optoelectronics, photonics, catalysis,imaging technology and drug delivery (Sharma et al., 2012).

4.3. Micronutrition

Gold is generally believed to be non-essential for microbial nutrition(Reith et al., 2007a). However, in the presence of gold,Micrococcus (M.)luteus produces an gold-containing protein, which oxidises methane tomethanol. The protein has a Au+/Au3+ redox couple in its active centreand presumably helps the bacterium to survive when usual sources ofcarbon and energy are scarce (Levchenko et al., 2000, 2002).M. luteusis amesophilic, aerobic and heterotrophic bacterium, which grows opti-mally near neutral pH values (Wieser et al., 2002). Methanotrophs havebeen detected on secondary gold grains from an Australian mine, sug-gesting an environmental association of methane-oxidising bacteriawith gold (Reith et al., 2007a). It is not known if other gold-containingenzymes exist in any other microorganisms (Reith et al., 2007a).

4.4. Ligand utilisation and loss

Microorganisms can decrease gold solubility by consuming theligands that bind gold. The ability of microorganisms to destabilisegold ligands will need to be controlled if biogenic or other alternativelixiviants are to be used for gold leaching.

4.4.1. Biooxidation and reductionThe precipitation of gold fromAu(I)-thiosulphate solutions has been

observed in the presence of thiosulphate-oxidising bacteria (Lengkeand Southam, 2005). Gold precipitated by Acidithiobacillus thiooxidanswas accumulated inside the bacterial cells as ne-grained colloids (510 nm in diameter) and in the bulk liquid as crystalline micrometer-scale gold. While gold was deposited throughout the cell, it was con-

centrated along the cytoplasmic membrane, suggesting that goldprecipitation was likely enhanced via electron transport processesassociated with energy generation (Lengke and Southam, 2005).

SRB, such as Desulfovibrio spp., typically oxidise organic compoundsusing sulphate as the terminal electron acceptor. Many SRB can also usealternative electron acceptors, such as thiosulphate. SRB have beenshown to reduce the thiosulphate from Au(I)-thiosulphate complexesand thus precipitate gold (Lengke and Southam, 2006). Lengke andSoutham (2007) studied the role of SRB in the precipitation of elementalgold using column experiments. Bacterially mediated gold precipitationfrom the Au(I)-thiosulphate complex was more efcient (98.299.6%)than the precipitation in corresponding abiotic controls (074.3%).SRB reduce thiosulphate and other sulphur compounds to H2S, whichprecipitates metals ions, such as Fe2+, leading to the formationof metal sulphides. Thus, SRB may also indirectly contribute to goldprecipitation by production of H2S which precipitates gold as sulphide,and iron sulphides which may lead to reduction of Au+ to Au0

(Lengke and Southam, 2006). The destabilised goldmay be incorporatedinto the newly forming sulphide minerals (Reith et al., 2007a).

Similarly to the thiosulphate-ligand utilisation, microbes utilisinggold-complexing carboxylic acids (such as amino-acids) or cyanidemay lead to destabilisation of gold complexes and contribute to goldprecipitation (Reith et al., 2007a). High pH has been suggested as anapproach to control cyanide degrading microbial populations (Fedel-Moen et al., 2000).

4.4.2. Sorption and accumulation of iodineIodine is a biophilic element, and accumulates in various organisms.

To date, however, themechanisms of iodine uptake by living organismshave been poorly understood with the exceptions of brown algae andthe thyroid gland in mammals (Amachi, 2008). Amachi et al. (2005a)isolated a marine bacterium Arenibacter sp. C-21, which can activelytransport and accumulate iodine. When grown in a liquid mediumcontaining 0.1 M I, 7989% of the Iwas removed from themedium,and a corresponding amount of I was detected in the cells. Whenthe strain was cultured with 0.1 M I, the maximum I content was220 3.6 pmol of I permg of dry cells, and themaximum concentra-tion factor for Iwas 5.5 103. In the presence of much higher concen-trations of I (1 M to 1 mM), I content increased, but decreasedconcentration factors for I were observed (Amachi et al., 2005a).Iodine transport assays revealed that glucose and oxygenwere necessaryfor the uptake of iodine (Amachi et al., 2007b). IO3, which is the otherdominant species of iodine in terrestrial and marine environments, wasnot transported (Amachi et al., 2005a).

In terrestrial environments, iodine is strongly adsorbed by soils(Amachi, 2008). Although the sorption by soils is affected by variousphysico-chemical parameters including soil type, pH, Eh, salinity, andorganic matter content, a number of studies have indicated that micro-organisms are also involved in the process (Amachi, 2008). Muramatsuand Yoshida (1999) showed that autoclaving soils signicantly reducedthe sorption of I and the sorption was recovered after adding a smallamount (110%) of fresh soil, suggesting the role of microorganisms insorption. Koch et al. (1989) observed increased I sorption in soilssupplemented with glucose and decreased sorption in soils treatedwith the antiseptic, thymol. Fumigation, air-drying and gamma-irradiation have also been shown to decrease iodine sorption(Bors and Martens, 1992). Iodine sorption in soils occurs moreeasily under oxic than under anoxic conditions (Bird and Schwartz,1996).

4.4.3. Volatilisation of iodineIodine is thought to volatilise in the form of organic iodine

compounds, such as methyl iodide (CH3I), diiodomethane (CH2I2),chloroiodomethane (CH2ClI) (Amachi, 2008). A wide variety ofmarine and terrestrial bacteria have been shown to be capable ofmethylating I to form CH3I. Aerobic bacteria such as Variovorax

sp. strain MRCD 30 and Rhizobium sp. strain MRCD 19 showed

81A.H. Kaksonen et al. / Hydrometallurgy 142 (2014) 7083considerable production of CH3I, whereas anaerobic Clostridium novyiand methanogens (Methanobacterium formicicum, Methanoculleusbourgensis, Methanosarcina mazei, and Methanospirillum hungatei) didnot produce CH3I (Amachi et al., 2001; Asakawa and Nagaoka, 2003).By using resting cells of a terrestrial bacterium Rhizobium sp. strainMRCD 19 and a marine bacterium Alteromonas macleodii strain IAM12920, Amachi et al. (2001) measured CH3I production at I2 concentra-tions of 0.1 mM to 5 mM. These strains showed increased CH3I produc-tion in the presence of increased I2 concentrations, indicating thatbacterial CH3I production depends greatly on the surrounding iodineconcentrations. The greatest observed production rate with Rhizobiumsp. was approximately 107 fmol CH3I day1 1010 cells1. Heat treated(80 C) or autoclaved cells of Rhizobium sp. strain MRCD 19 did notshow any CH3I production, suggesting that the methylation was medi-ated by live cells (Amachi et al., 2001). The addition of yeast extractand glucose to soil slurries stimulated iodine volatilisation, whereasautoclaving and addition of antibiotics (streptomycin and tetracycline,specic inhibitors of prokaryotes) decreased the volatilisation(Amachi et al., 2003). Along with biosorption and bioaccumulation,the volatilisation of iodide can lead to the loss of the lixiviant ifiodideiodine system is used for gold leaching.

5. Conclusions

Microorganisms play many roles in the biogeochemical cyclingof gold and can be utilised in a number of ways for gold processingand recovery. Biooxidation of refractory gold bearing sulphide oreswith acidophilic iron and sulphur-oxidising microorganisms has beenalready commercially practised since the 1980s, rst in bioreactorsand subsequently as heap leaching operations. Although in situ or inplace biooxidation of gold ores has not yet been industrially practised,the indirect oxidation of the ores with Fe3+ biologically regeneratedabove groundmay be feasible. However, research is still needed to dem-onstrate practical applicability and economic feasibility of the concept.Microbial processes can also be used to deactivate carbonaceous mate-rials that would bind solubilised gold from pregnant leach solutions.Mechanisms for biological permeability enhancement have also beenidentied. However, these processes may be slow and the feasibility ofin situ permeability enhancement of ore bodies is yet to be demonstratedthrough research.

Microorganisms can promote gold solubilisation by the excretion ofligands, such as thiosulphate, organic acids, cyanide and iodideiodine,which oxidise and forms complexes with gold. As thiosulphate, organicacids and cyanide are intermediates inmicrobial metabolism, their longterm stability in anunderground environmentmay beproblematic. Theselimitationsmayhinder theutilisation ofmicrobial gold solubilisation in anindustrial process. The microbial stability of chemically lixiviants, suchas thiosulphate and iodideiodine also needs to be considered in longterm industrial application.

Microorganisms can contribute to the recovery and/or loss of gold bydecreasing the solubility of gold through biosorption and accumu-lation, reductive precipitation, ligand utilisation and by using goldas a micronutrient for enzymes. Bioprecipitation and biorecoveryof gold would need to become economically attractive to be ableto replace already proven recovery processes such as activated car-bon adsorption. Future research efforts are needed to quantify theextent of gold loss from alternative lixiviant systems by microbialactivities.

In summary, several microbial processes are relevant for goldleaching and recovery. Some of these may be applicable for in situ orin place leaching of low grade gold ores, which the industry will in-creasingly depend on into the future. Other microbial processesneed to be considered as potential risks for lixiviant stability andgold recovery. The understanding of these processes will enhanceindustrial applications of biotechnology and lixiviant use by the

minerals industry.Acknowledgements

The support of the CSIRO Minerals Down Under National ResearchFlagship, sponsors of MERIWA project M409 and ORICA is gratefullyacknowledged.

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